Isoprenoid Biosynthesis in Pathogenic Bacteria: Nuclear Resonance Vibrational Spectroscopy Provides Insight into the Unusual [4Fe‐4S] Cluster of the E. coli LytB/IspH Protein

Abstract

The LytB/IspH protein catalyzes the last step of the methylerythritol phosphate (MEP) pathway which is used for the biosynthesis of essential terpenoids in most pathogenic bacteria. Therefore, the MEP pathway is a target for the development of new antimicrobial agents as it is essential for microorganisms, yet absent in humans. Substrate‐free LytB has a special [4Fe‐4S]2+ cluster with a yet unsolved structure. This motivated us to use synchrotron‐based nuclear resonance vibrational spectroscopy (NRVS) in combination with quantum chemical‐molecular mechanical (QM/MM) calculations to gain more insight into the structure of substrate‐free LytB. The apical iron atom of the [4Fe‐4S]2+ is clearly linked to three water molecules. We additionally present NRVS data of LytB bound to its natural substrate, (E)‐4‐hydroxy‐3‐methylbut‐2‐en‐1‐yl diphosphate (HMBPP) and to the inhibitors (E)‐4‐amino‐3‐methylbut‐2‐en‐1‐yl diphosphate and (E)‐4‐mercapto‐3‐methylbut‐2‐en‐1‐yl diphosphate.

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

“…[18–21] For example, the NRVS spectrum of oxidized Pyrococcus furiosus ferredoxin containing a D14C mutation (which therefore has 4 Cys ligands; Figure 1B, black line) [20] , is very similar to that seen with the model compound [Fe 4 S 4 Cl 4 ](Ph 4 P) 2 , which contains a [Fe 4 S 4 Cl 4 ] 2− cluster in which the four terminal ligands are Cl (Figure 1B, grey line) [20] . Basically the same features are also seen in oxidized IspH with HMBPP ( 2 ), the amino-analog ( 6 ) or the thiol analog ( 7 ) as ligands [16] , in which O, N or S are directly bonded to the 4 th Fe. In sharp contrast, these features are all less obvious (or absent) in the NRVS spectrum of IspH in the absence of 2 , 6 or 7 [16] .…”

“…However, the results of 57 Fe Mössbauer spectroscopy [15] indicated the presence of a 5 or 6-coordinate 4th iron in “ligand-free” oxidized IspH, with three cluster S and most likely three additional N/O ligands bound to the 4 th Fe. More recently, Faus et al [16] reported a NRVS (nuclear resonant vibrational spectroscopy) investigation of oxidized IspH and suggested that the three non-cluster ligands were H 2 O molecules (or presumably OH − , or a mixture of both). This structure is surprisingly labile, leading under crystallization conditions to loss of the 4 th Fe.…”

COST Action CM1201 “Biomimetic Radical Chemistry”: free radical chemistry successfully meets many disciplines

“…Thus, hexane is oxidized by the proteobacterium strain HxN1 in a tandem process requiring two radical enzymes: the first of which attacks a C-H bond, whilst the second effects a coenzyme B 12 -dependent molecular rearrangement. A putative HxN1 enzyme uses a cysteinyl radical to abstract stereospecifically the pro-S hydrogen atom from C-2 of hexane.…”

“…Computational chemistry employing density functional theory supported this concept in an inter-Working Group collaboration that will be published in due time [L. A. Eriksson (WG2), Buckel, B. T. Golding, and D. M. Smith (Zagreb)]. Current work is focused on the analogous degradation of decane and defining the role of coenzyme B 12 in alkane degradation. The expertise of I. Smonou's group in stereochemically controlled enzymatic reductions of ketones [4] led, in collaboration with Golding's group, to the synthesis of (2R,9S)-decane-2,9-diol, a precursor of (2R,9S)-[2,9-2 H 2 ]decane, required for studies of anaerobic decane oxidation.…”

“…IspH/LytB, the last enzyme of the methylerythritol phosphate pathway, converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In collaboration with V. Sch€ unemann (TU Kaiserslautern), the structure of substrate-free LytB was clarified in solution [12]. Potent LytB inhibitors were designed and prepared in collaboration with D. E. Poulter (University of Utah, Salt Lake City, UT, USA), whereby the OH group of HMBPP was replaced by a thiol (TMBPP, K i 20 nM) or an amino group (AMBPP, K i 54 nM) [13].…”